All eyes show an association of photoreceptors with dark pigment. In vertebrates, this association can be traced back to the actions of the bHLH-Zip transcription factor MITF, which controls the generation of melanin-bearing pigment cells, and the paired-domain transcription factor PAX6, which controls the generation of photoreceptors in the retina. High level expression of these transcription factors is usually mutually exclusive, except in the neuroepithelium-derived retinal pigment epithelium (RPE) where MITF and PAX6 are co-expressed during development. We approached the question of the role of PAX6 in the RPE genetically, using a variety of mouse Mitf and Pax6 alleles individually and in combinations. The results show that a reduction in the level of functional PAX6 protein exacerbates the dorsal RPE-to-retina transition normally associated with Mitf mutations, and that overexpression of PAX6 protein alleviates this Mitf-mediated RPE-to-retina transition. In fact, candidate gene expression analyses and gene expression profiling indicate that in the RPE, Pax6 normally reduces the expression of pro-retinogenic genes, while in the retina, it promotes their expression. Given that Pax6 plays important roles in the development of cornea, lens, retina and now RPE, we conclude that it is involved in the development of all major structures of the vertebrate eye. Its expression, however, does not extend towards the developing eyes'connection to the brain, called the optic stalk, where its expression is in part suppressed by the homeodomain transcription factors VAX1/2. However, the combination of mutations in Vax1 and Vax2 does not only lead to a de-repression of Pax6 but also a de-repression of Mitf and, consequently, a conversion of the dorsal optic stalk into an RPE. Hence, during eye development, PAX6, MITF, and VAX1/2 are crucial to set up the correct boundaries between retina and RPE and between RPE and optic stalk. Current studies aimed at understanding the set of target genes activated by the respective transcription factors will eventually give us insights into abnormal eye development and disease.